Project Summary Human skin equivalents (HSEs) have provided an effective therapy for patients with significant skin loss due to burns, ulcers and genetic skin diseases; however they still have various limitations including poor viability and lack of appendages or mismatch in hair density. We have recently improved the viability of skin grafts by establishing a method to micropattern vasculature in HSEs. On the other hand, it still remains a prevailing challenge to engineer functional skin grafts with hair follicles since long-term cultured human dermal papilla cells (DPCs) in vitro lose their hair-inducing capacity. The overall goal of this project is to engineer viable and functional skin equivalents with appendages using cultured human cells. We will employ bioengineering, genetics and systems biology approaches to reprogram cultured DPCs and dermal fibroblasts into hair inductive cells. These cells will then be used to generate hair-bearing 3D skin equivalents, enabling the development of truly functional skin substitutes for patients with significant skin/hair loss. In particular, we will use i) microfabrication techniques to recapitulate 3D hair follicle microenvironment (microenvironmental approach) and ii) reverse engineering gene network analysis to reprogram hair inductive gene signatures of cultured DPCs and fibroblasts (genetic approach). Finally, taking advantage of our recently established technique for vascularization, we will develop viable HSEs that, after engraftment, can controllably regenerate hair and remain fully integrated due to enhanced revascularization. The ability to regenerate an entire hair follicle from cultured human cells will have an overwhelming positive impact on the medical management of different types of alopecia, epidermolysis bullosa as well as chronic wounds, severe infections, and burns, all of which represent major unmet medical needs. The ability to generate a hair-bearing skin is a crucial step towards making a truly functional human skin and would mark a dramatic conceptual advance in regenerative medicine approaches to disorders of the skin and hair follicle, such as hair loss, as well as improve the outcome of severe skin injuries leading to disfiguring scars.